This invention relates to calorimetric detection of analytes in a liquid sample, and finds application in the fields of biology, medical analysis, and analytical chemistry.
This section discusses a variety of methods for detection of compounds by the appearance of color. However, the citation of a reference or concept in this section should not be construed as an indication that the reference or concept is prior art to the present invention.
Detecting and measuring color is a convenient method for measuring the amount of a substance in solution. If the substance to be detected, i.e., the xe2x80x9canalytexe2x80x9d, does not have an inherent color, a color may be produced, as surrogate for the substance, by a variety of chemical, enzymatic or immunochemical methods. This well known art is practiced, for example, in both research and clinical laboratories of biological and health care fields. The principle of colorimetry is the constant loss of light in passage through a solution by absorbance of light into the colored compound. A molecular species absorbs the same amount of light in proportion to its concentration at the same wavelength every time it is measured. Light lost as it passes through a solution is determined by the concentration of the absorbing molecules and the length of the light path. By knowing the length of the light path and the light loss and the volume of the solution, it is possible to calculate the amount, or mass, of a substance. In practice, the calculation is frequently replaced by a standard curve represented by the same reaction on a series of known amounts of the same substance that have been processed by the same reactions to produce color. In alternative methods, the amount of light lost by scattering can be determined.
Ligand Assay
The sensitivity of a colorimetric test is defined as the limit amount that may be detected reliably using the method. One way to increase sensitivity (i.e., lower the limit amount to be detected), is to use an amplifier method for producing color. Amplifiers of special interest to life science assay design are catalysts, and especially the class of biological catalysts known as enzymes. Color reactions for the detection of enzymes or for the detection of the substrates on which enzymes operate as catalysts are well known. Sensitivity may be improved further by attaching enzymes to molecules that recognize the analyte, usually referred to as ligands.
ELISA
One standard assay for detecting and quantifying an analyte in a solution is the Enzyme Linked Immunosorbent Assay, or ELISA. However, this assay can be difficult to carry out and expensive. In this assay, after the enzyme linked ligand is joined to all analyte present in the assay, the excess enzyme linked ligand that is not attached to analyte must be eliminated from the solution or it produces unwanted amplification signal. The standard assay design includes a series of steps as follows:
One example of a standard container for such assays is a 96 well plate, so called because it has a matrix of 12 by 8 wells of standard size in a standard size frame. Manufacturers may treat such plates so as to permit strong attachment of certain molecular species to the well walls.
Adding a solution containing ligand to each well on the plate at 4xc2x0 C. permits attachment and retains activity of ligand. This is an xe2x80x9covernightxe2x80x9d procedure.
Plates are then washed to eliminate any ligand not firmly attached to the well wall. There are three sequential washes.
Plates are most often used immediately because the ligands so attached are not stable to storage. Sample or standard analyte solutions or blanks containing reagent only are added to individual wells. The analyte species, which forms the ligand pair, attaches to the ligand on the well wall.
After some period of incubation the plates are again washed to eliminate the remaining sample solution. There are three sequential washes
Enzyme-conjugated ligand is then added to each well. Usually this ligand has specificity for another recognition site on the analyte molecule.
The plate is again washed to eliminate excess enzyme-conjugated ligand remaining in solution. There are three sequential washes. At this point the amount of enzyme attached to the wall of each well is determined by the amount of analyte also attached to the well wall.
Reagents are then added to test for the presence of the enzyme, and the color so produced relates to the amount of analyte added to each well.
Measurement is made in a photometer designed for reading the plates, called a plate reader.
It is apparent that ELISA is a tedious, time-consuming assay with many necessary steps. A manufacturer may perform the initial preparation of plates. Such manufactured plates are expensive. For example, one assay plate for performing 96 tests, may cost $650. In use, the assay using such a plate still takes approximately 5 hours to complete.
Channeling
One improvement over ELISA methods is known as xe2x80x9cchannelingxe2x80x9d described in Gibbons et al., Methods of Enzymology 136:93. The principle of channeling is to form small, specialized particulates during the assay. The particulates permit attachment of ligands with two separate enzymes. The enzymes act in coordination, such that the product of one enzyme acts as substrate for the next. Only enzymes attached to the particles permit channeling. Enzyme not attached to particles do not produce color reaction product. Although this is a theoretical improvement over ELISA, there are considerations in formation of such specialized particles, which make this design impractical.
Other Solution Assays
Other solution assays using amplifiers are known. For example, U.S. Pat. No. 3,975,237 discloses a solution assay typically for small molecules. The assay principle is an inhibition of enzyme activity by use of a large molecule receptor, for example an antibody to the small molecular weight analyte, as competition to a small molecular version of the same analyte molecule. Methods of preparing conjugates of enzymes with analytes, and the sensitivity of assays of this nature are described. Another solution assay is described by Kricka and Ji, 1994, Clinical Chemistry 40:1828 -30. This assay uses small molecule aryl boronic acids to enhance enzymatic luminescence. In a similar assay described in U.S. Pat. Nos. 5,843,666 and 5,306,621, the chemiluminescence is further enhanced by small molecule phenols. The method uses a binding partner labeled with a hydrolytic enzyme to produce a phenolic enhancer in close proximity to a peroxidase labeled specific binding partner. The mechanism of the enhancement is not known. This is a luminescence assay that uses expensive equipment that is not available to large numbers of laboratories, and has limited sensitivity.
Histochemistry
In the fields of histochemistry and cytochemistry, color contrast in tissues or cells is produced for purpose of microscopic examination or detection. Ligands that recognize tissue components and conjugated to enzymes are used as amplifiers that may then produce color with appropriate reagents. For visual examination it is possible to provide several colors of reaction product for several individual analytes with several different enzyme-conjugated ligands. This method is acceptable as long as the different analytes are located in different cells or tissue components. However, if the two analytes are present in the same location the resulting colors are additive, producing a new color which cannot be interpreted by microscopy. Another way to resolve two colors in one location is to use fluorescent markers. However fluorescence microscopy is much more expensive, and the automatic detection of fluorescence requires longer integration times, thus making automation of two color image detection impractical.
Color Photographic Development
Exposure of color film to light produces activated silver granules in the film. Light of different colors activates silver particles in different layers of the color film. During development with a common reagent, silver grains are reduced and thereby oxidize the common developer. The oxidized developer is captured in the layer chemically combines with color couplers. It is only the product of coupling oxidized developer and color coupler that produces color. Each layer has at least one coupler producing a color reaction product specific for that layer. Scavenger molecules, sometimes called xe2x80x9cwhite couplers,xe2x80x9d prevent diffusion of developer from one layer to another. It is considered an advantage of the reaction of scavengers and colored couplers with oxidized developers if the reaction product is retained in the layer where it is formed. This is accomplished by designing or selecting couplers that are insoluble in the developer solvent both before and after coupling takes place. Some scavengers and some color couplers are designed with attachment to immobilized polymers. There are active regions on color coupler molecules and white couplers which enhance coupling to oxidized or activated photographic developers.
The chemical structure of the color couplers is highly similar to the reaction product of histochemical color producing compounds. Indeed the histochemical and cytochemical substrates are often the same as photographic color couplers with addition of a protective group on the active site. The protective group is hydrolyzed from the active region by action of the enzyme of interest. The chemical structure of photographic color developers is also very similar to substrates used in histochemistry of peroxidase reactions. The oxidized reaction product is also similar in histochemistry and in color photographic developers. In both chemical reactions the oxidation potential is also approximately the same. Use of photographic developers in detection of reaction products of hydrolytic enzymes was suggested by, for example Ornstein, 1959, Histochemistry and Cytochemistry, 7: 231 and Ornstein, 1974, Histochemistry and Cytochemistry 22:453-69, both incorporated by reference herein.
In the photographic industry it is well known that certain couplers form color more efficiently than others. By this is meant that they require less or more oxidized developer to form color. Since the amount of oxidized developer is determined by the number of sensitized silver ions, the effect is to require more sensitized silver ions for some couplers than for others. The more efficient couplers are known as 2-equivalent couplers, while the less efficient couplers are known as 4-equivalent couplers.
The present invention provides an assay for analytes in solution. The assay relies, in part, on associating two catalytic activities in close proximity to each other to produce a detectable product. The invention takes advantage of the proximity of two catalytic activities, for example enzyme activities, to limit production of color, while the same enzymes not in proximity produce only minor background color. The present invention makes use of compounds analogous to protected color couplers and analogs to photographic color developers to produce color that is limited to regions in the solution where an oxidizing enzyme and a hydrolytic enzyme are in proximal location. In one embodiment of the invention white couplers are used as scavengers to inhibit color production in the solution where the enzymes are not in proximity.
The present invention has advantages over existing methods for detecting analytes in solution, such as ELISA. For example, the present assay can be carried out in multi-well (e.g., 96-well) plates without any wash steps to remove non-bound ligand, non-analyte molecules in the sample, and non-bound enzyme conjugated ligand. Without any attachment of capture reagent, analyte or detection reagent to the well wall, the present invention permits completion of a test within 1 to 1.5 hours. The results can be read using colorimetry plate readers, widely available clinical and research laboratories. Thus, the assay of the present invention is more rapid, has fewer steps, and is more economical than existing methods.
In one aspect, the invention provides a method for detecting an analyte in solution comprising (a) combining (i) a solution to be assayed for the presence or amount of the analyte; (ii) a first ligand capable of binding the analyte, wherein said first ligand is directly or indirectly bound to a first enzyme capable of cleaving a first substrate to produce a colorless first product, wherein said first enzyme is a hydrolase; (iii) a second ligand that binds the analyte, wherein the binding of the first ligand to the analyte does not interfere with the binding of the second ligand, and wherein the second ligand is directly or indirectly bound to a second enzyme capable of oxidizing a second substrate to produce a colorless second product, wherein said second enzyme is an oxidase; (iv) said first substrate; and, (v) said second substrate; whereby the hydrolase cleaves the first substrate to product the first product and the oxidase oxidizes that second substrate to produce the second product, wherein the first product and the second product chemically combine to produce a detectable reaction product, said detectable reaction product being a colored reaction product; (b) detecting the production of the colored reaction product; (c) relating the production of the colored reaction product with the presence of analyte in the solution. The method can further comprise combining a compound that is a scavenger for the first reaction product or the second reaction product in step (a). The scavenger can be 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide. The first substrate can be a compound that comprises a benzene ring or naphthalene structure with one active hydroxyl group, e.g., 1-naphthol phosphate or phenyl phosphate. The second substrate can be N,N-dimethyl paraphenylene diamine; N,N-diethyl paraphenylene diamine; N-phenyl paraphenylene diamine; Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylene diamine; 4 amino antipyrene; or N,N-dimethylamino benzidine. In various embodiments, the first ligand is a first antibody that specifically binds the analyte and second ligand is a second antibody that specifically bind the analyte, the hydrolase is a phosphatase, an esterases, a galactosidase, a lipase, a glucuronidase, an amidase, a peptidase, or a sulphatase. In an embodiment, for example the hydrolase is alkaline phosphatase and the oxidase is horseradish peroxidase. In an embodiment, the first substrate is naphthyl phosphate or phenyl phosphate and the second substrate is Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine. In some embodiments, at least one of the first and second ligand is an antibody or a lectin.
In a related aspect, the invention provides a method for detecting an analyte in solution comprising (a) combining (i) a solution to be assayed for the presence or amount of the analyte, wherein said analyte has an oxidase activity capable of acting on a first substrate to produce a colorless first product; (ii) a ligand capable of binding the analyte, wherein said ligand is directly or indirectly bound to a first enzyme capable of cleaving a second substrate to produce a colorless second product, wherein said first enzyme is a hydrolase; (iii) said first substrate; and, (v) said second substrate; whereby the hydrolase cleaves the second substrate to product the second product and the oxidase oxidizes the first substrate to produce the first product, wherein the first product and the second product chemically combine to produce a detectable reaction product, said detectable reaction product being a colored reaction product; (b) detecting the production of the colored reaction product; (c) relating the production of the colored reaction product with the presence of analyte in the solution. In an embodiment, the analyte has a pseudoperoxidase activity. In an embodiment, the analyte is glycated hemoglobin. In an embodiment, the solution comprises non glycated hemoglobin and the glycated portion of hemoglobin to be compared to total hemoglobin. In various embodiments, the ligand is an organic boronic acid compound directly or indirectly conjugated to a hydrolase. Further, the hydrolase can be alkaline phosphatase; the method can include combining a compound that is a scavenger for the first reaction product in step (a); the first substrate is selected from the group N,N-dimethyl paraphenylene diamine; N,N-diethyl paraphenylene diamine; N-phenyl paraphenylene diamine; Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylene diamine; 4 amino antipyrene; and N,N-dimethylamino benzidine, the second substate is naphthyl phosphate or phenyl phosphate, and the scavanger is 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide.
In a related aspect, the invention provides a method for detecting an analyte in solution comprising (a) combining (i) a solution to be assayed for the presence or amount of the analyte, wherein said analyte has a hydrolase activity capable of acting on a first substrate to produce a colorless first product; (ii) a ligand capable of binding the analyte, wherein said ligand is directly or indirectly bound to a first enzyme capable of cleaving a second substrate to produce a colorless second product, wherein said first enzyme is a oxidase; (iii) said first substrate; and, (v) said second substrate; whereby the hydrolase cleaves the first substrate to produce the first product and the oxidase oxidizes the second substrate to produce the second product, wherein the first product and the second product chemically combine to produce a detectable reaction product, said detectable reaction product being a colored reaction product; (b) detecting the production of the colored reaction product; (c) relating the production of the colored reaction product with the presence of analyte in the solution.
Stated differently, in various aspects and embodiment, the invention provides (1) A quantitative or qualitative colorimetric solution assay for analytes comprising: providing an analyte in solution; providing a first ligand to the analyte; providing a second ligand to the analyte; providing a catalytic activity for the first ligand to the analyte; providing a different catalytic activity for the second ligand to the analyte; providing a reagent for the first catalytic activity devised to give a first colorless reaction product; providing a reagent for the second catalytic activity devised to give a second colorless reaction product; devising conditions where the further reaction of the first reaction product and the second reaction product produces a colored third reaction product only when the first ligand and the second ligand are attached to the same analyte molecule; detecting the third reaction product by the amount of color produced and relating the detected color to the analyte in solution. In various embodiments: a reagent acting as a scavenger for the first or second reaction product is provided; the catalyst attached to the first ligand is an enzyme, such as an oxidase (e.g., horseradish peroxidase); the catalyst attached to the second ligand is an hydrolase enzyme (e.g., alkaline phosphatase); the second ligand is attached to a different epitope or attachment than the first ligand; first catalyst is horseradish peroxidase, the second catalyst is alkaline phosphatase, the first reagent is an oxidizable developer and the second reagent is naphthyl phosphate or phenyl phosphate; the oxidisable developer is Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; the reaction comprises scavenger (e.g., 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide).
In various embodiment, at least one ligand is an antibody; or at least one ligand is a lectin; or at least one of the ligands is a molecule with more general affinity properties (e.g., a boronic acid compound)
In one aspect, the invention provides: a quantitative or qualitative colorimetric solution assay for analytes comprising: providing an analyte with a first catalytic activity in solution; providing a ligand to the analyte; providing a second catalytic activity for the ligand to the analyte; providing a reagent for the first catalytic activity devised to give a first colorless reaction product; providing a reagent for the second catalytic activity devised to give a second colorless reaction product; devising conditions where the further reaction of the first reaction product and the second reaction product produces a colored third reaction product only when the ligand is attached to the analyte; detecting the third reaction product by the amount of color produced and relating the detected color to the analyte in solution.
In various embodiments, a scavenger for the first or second reaction product is included, the catalyst analyte is an enzyme or pseudoenzyme; the enzyme is an oxidase (e.g., peroxidase or pseudoperoxidase) the catalyst attached to the ligand is a hydrolase enzyme (e.g., alkaline phosphatase); the ligand is attached to a different epitope or attachment than the enzyme or active site of the analyte; the first catalyst is a peroxidase, the second catalyst is alkaline phosphatase, the first reagent is an oxidizable developer and the second reagent is naphthyl phosphate or phenyl phosphate.
In various embodiments, the first catalyst is glycated hemoglobin; there is present in the analyte sample a quantity of non glycated hemoglobin and the result to be determined is the determination of the glycated portion of hemoglobin compared to total hemoglobin; the oxidizable developer is Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine; a scavenger reagent is included (e.g., 3-amino-1-(2,4,6-trichlorophenyl)-2-pyrazolin-5-one or acetoacetamide); the ligand is an antibody; the ligand is a lectin; the ligand is a molecule with more general affinity properties (e.g. a boronic acid compound, attached to alkaline phosphatase.
In a related aspect, the invention provides a kit for solution assays comprising a first antibody to an analyte conjugated to a first enzyme with peroxidase activity, a second antibody to the same analyte conjugated to a second enzyme with alkaline phosphatase activity, a source of hydrogen peroxide, an oxidizable developer, a phenol like substrate for alkaline phosphatase and a colorless coupler to use as scavenger. In an embodiment, the oxidizable developer is Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine, the substrate is phenyl phosphate or naphthyl phosphate and the scavenger is acetoacetamide. The invention also provides a kit for detecting the proportion of glycated to total hemoglobin comprising a well plate suitable for measurement in a plate reader, an enzyme with alkaline phosphatase activity coupled with a boronic acid, a source of hydrogen peroxide, an oxidizable developer, a phenol like substrate for alkaline phosphatase and a colorless coupler to use as scavenger. In an embodiment, the oxidizable developer is Nxe2x80x2-ethyl-Nxe2x80x2ethyl-(2-methylsulfonamidoethyl)-2-methyl-1,4-phenylenediamine, the substrate is naphthyl phosphate and the scavenger is acetoacetamide.